Loss of CRWN Nuclear Proteins Induces Cell Death and ... · nents, based on pioneering studies by...

Loss of CRWN Nuclear Proteins Induces Cell Death andSalicylic Acid Defense Signaling1[OPEN]

Junsik Choi,a,b Susan R. Strickler,b and Eric J. Richardsb,2,3

aSection of Plant Biology, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853bBoyce Thompson Institute, Ithaca, New York 14853

ORCID IDs: 0000-0002-7122-3290 (J.C.); 0000-0002-0121-0048 (S.R.S.); 0000-0002-8665-7470 (E.J.R.).

Defects in the nuclear lamina of animal cell nuclei have dramatic effects on nuclear structure and gene expression as well asdiverse physiological manifestations. We report that deficiencies in CROWDED NUCLEI (CRWN), which are candidate nuclearlamina proteins in Arabidopsis (Arabidopsis thaliana), trigger widespread changes in transcript levels and whole-plantphenotypes, including dwarfing and spontaneous cell death lesions. These phenotypes are caused in part by ectopicinduction of plant defense responses via the salicylic acid pathway. Loss of CRWN proteins induces the expression of thesalicylic acid biosynthetic gene ISOCHORISMATE SYNTHASE1, which leads to spontaneous defense responses in crwn1 crwn2and crwn1 crwn4 mutants, which are deficient in two of the four CRWN paralogs. The symptoms of ectopic defense response,including pathogenesis marker gene expression and cell death, increase in older crwn double mutants. These age-dependenteffects are postulated to reflect an increase in nuclear dysfunction or damage over time, a phenomenon reminiscent of agingeffects seen in animal nuclei and in some human laminopathy patients.

The morphology of cellular organelles is closely re-lated to their function and physiological state. For ex-ample, mitochondrial cristae result from a complexcorrugation of the inner membrane required to ac-commodate enzymes for cellular respiration. Mito-chondria in skeletal or heart muscle, which requirehigh amounts of energy, have stacked cristae, whiletissues with lower energy demands, such as liver orkidney, contain mitochondria with less stacked cris-tae (Kühlbrandt, 2015). This relationship implies thatstructural components, which provide physical sup-port and define organellar structure, are intimatelyinvolved in organelle function. In nuclei, the relation-ship between morphology and function is particularlyimportant, as nuclei possess genetic information whoseexpression can be influenced by changes in nuclear

structure and organization (Reddy et al., 2008; Schreiberand Kennedy, 2013; Davidson and Lammerding, 2014).Eukaryotic nuclei are wrapped in a nuclear envelope,

a double leaflet consisting of outer and inner nuclearmembranes. There are various proteins associated withthe envelope essential for nuclear function and mor-phology. Examples include nucleoporins, which formnuclear pores, Linker of Nucleoskeleton and Cytoskel-eton complex proteins, which span the nuclear enve-lope (Lombardi and Lammerding, 2011; Tapley andStarr, 2013; Tatout et al., 2014; Meier, 2016), and a nu-clear lamina (NL) structure underlying the inner nu-clear envelope (Aebi et al., 1986). Among these, the NLis a key architectural feature that affects both the mor-phology and the function of nuclei. In animals, theNL isa reticular structure under the inner nuclear membrane.The animal NL is mainly composed of intermediatefilament-like proteins called lamins, which polym-erize to form fibrillar networks (Aebi et al., 1986).This structure provides docking sites for chromatinand serves as a physical support for the organelle(Gruenbaum and Foisner, 2015). Most heterochroma-tin resides near or at the nuclear periphery, which istypically a repressive environment for gene expression(Egecioglu and Brickner, 2011). Studies have demon-strated that tethering genes to the NL results in tran-scriptional repression in mammalian cells (Reddyet al., 2008). Genomic profiling approaches have de-fined lamina-associated domains (LADs), character-ized by repressive chromatin and genes with lowexpression levels (Pickersgill et al., 2006; Guelen et al.,2008; Ikegami et al., 2010; Peric-Hupkes et al., 2010;van Steensel and Belmont, 2017). This interaction be-tween the genome and NL can link alterations in NL

1This work was supported by the facilities and financial support ofthe Boyce Thompson Institute. Partial funding for this project wasderived fromU.S. National Science Foundation grants to E.J.R. (MCB-0956820 and MCB-1158297). J.C. was funded in part by the KoreanGovernment Scholarship Program for Study Overseas and CornellUniversity.

2Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Eric J. Richards ([email protected]).

J.C. and E.J.R. conceived and designed the experimental plan; J.C.performed most of the experiments; S.R.S. contributed to the bioin-formatics analysis; E.J.R. supervised the experiments; J.C. and E.J.R.wrote the article.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.18.01020

Plant Physiology�, April 2019, Vol. 179, pp. 1315–1329, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 1315 www.plantphysiol.orgon October 26, 2020 - Published by Downloaded from

Copyright © 2019 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0002-7122-3290

http://orcid.org/0000-0002-7122-3290

http://orcid.org/0000-0002-0121-0048

http://orcid.org/0000-0002-0121-0048

http://orcid.org/0000-0002-8665-7470

http://orcid.org/0000-0002-8665-7470

http://orcid.org/0000-0002-7122-3290

http://orcid.org/0000-0002-0121-0048

http://orcid.org/0000-0002-8665-7470

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.18.01020&domain=pdf&date_stamp=2019-03-23

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.18.01020


structure to genomic instability as well as modifica-tions in nuclear morphology. In a well-documentedexample, certain dominant mutations in the humanlamin A gene lead to abnormal nuclear shape, chro-matin organization defects, and clinical syndromes, suchas premature aging (e.g. Hutchinson-Gilford progeriasyndrome; Hutchinson, 1886; Gilford, 1904; Goldmanet al., 2004). Also, fibroblasts cultured from Hutchinson-Gilford progeria syndrome patients exhibit repositionedchromosomes within the nucleus and transcriptionalmisregulation (Csoka et al., 2004; Meaburn et al., 2007).Thesefindings indicate that theNL is essential for propergene expression (Zheng et al., 2018).

Although plants lack intermediate filaments andlamin orthologs, different classes of nuclear coiled-coilproteins have been identified as putative NL compo-nents, based on pioneering studies by Masuda andcolleagues on carrot (Daucus carota) Nuclear MatrixConstituent Proteins (NMCPs; Masuda et al., 1993,1997). In Arabidopsis (Arabidopsis thaliana), NMCPorthologs, called CROWDED NUCLEI (CRWN) pro-teins, are candidates for the major structural compo-nent of a plant NL. The Arabidopsis genome containsfour CRWN genes, which are expressed broadly at thetranscript level without obvious tissue specificity. Theproteins partition into two distinct clades: one con-taining the CRWN1, CRWN2, and CRWN3 paralogs(NMCP1 clade), and a distinct one including CRWN4(NMCP2 clade). Among these paralogs, CRWN1 andCRWN4 proteins localize to the nuclear periphery andare therefore the best candidates for NL components,while CRWN2 and CRWN3 are distributed through-out the nucleoplasm (Dittmer et al., 2007; Dittmer andRichards, 2008; Sakamoto and Takagi, 2013). CRWNgenes are essential, as a loss of all four genes leads toinviability. CRWN proteins are important for main-tenance of nuclear structure andmorphology, as manycrwn mutants have small and round nuclei (Dittmeret al., 2007; Sakamoto and Takagi, 2013; Wang et al.,2013) compared with wild-type Arabidopsis nuclei,which are elongated in many differentiated cell types(Chytilova et al., 2000; Meier et al., 2016, 2017). Ourgroup previously reported that CRWN genes haveroles in specifying the structure of heterochromatinaggregates in interphase (chromocenters), implyingthat CRWN proteins not only control nuclear mor-phology but are also able to regulate higher ordergenome organization (Dittmer et al., 2007; Wang et al.,2013). Whole-plant phenotypes of crwn mutants showthat many double and triple crwn mutants are smallerin size and have wrinkled leaves, demonstrating thataltered plant nuclear structure can ultimately lead toabnormal growth.

Here, we investigate the effects of crwnmutations ongene expression and elucidate a mechanism throughwhich loss of CRWN proteins causes dwarfism. Weshow that mutants lacking the full complement ofCRWN genes show altered gene expression patterns,characterized by overexpression of transcripts associ-ated with biotic pathogen response. Plants lacking both

CRWN1 and CRWN2, or CRWN1 and CRWN4, exhibitectopic defense responses, including partial resistanceagainst the virulent bacterial pathogen, Pseudomonassyringae pv tomato (strain DC3000). In addition, thesecrwn genotypes exhibit spontaneous cell death,which is associated with the up-regulation of thesalicylic acid (SA)-biosynthesis gene, ISOCHORISMATESYNTHASE1 (ICS1/SID2), and subsequent SA-dependentdefense signaling. Our results demonstrate that nuclearperiphery defects in plants, like similar changes in animalcells, can lead to premature cell death.

RESULTS

Three crwn Single Mutants Show Altered TranscriptionalProfiles in the Absence of Whole-Plant Phenotypes

We reasoned that the alterations in nuclear size andshape in the crwn mutants could have an effect ontranscriptional regulation and/or posttranscriptionalprocessing. To test this prediction, we assessed the ef-fect of crwn mutations on steady-state transcript levelsusing RNA sequencing (RNA-seq). Four-week-old ro-sette leaves from five mutants (crwn1, crwn2, crwn4,crwn1 crwn2, and crwn1 crwn4) were used as tissue forthe RNA purification and transcriptomic analysis.These five mutants encompass the range of phenotypesdisplayed by crwn family mutants and involve both theNMCP1 and NMCP2 clades of CRWN paralogs. Mu-tants lacking either CRWN1 or CRWN4 show abnormalnuclei that fail to elongate in differentiated cells and arereduced in size. The crwn2 mutation by itself does notalter nuclear morphology, but it was included in ouranalysis because crwn1 crwn2 double mutants havemuch smaller nuclei and display dwarf phenotypes(Dittmer et al., 2007; Wang et al., 2013), indicating thatCRWN1 and CRWN2 have at least partially over-lapping function. Similarly, crwn1 crwn4 double mu-tants are smaller than the wild type or either crwn1 orcrwn4 single mutant.

We first compared misregulated genes among thethree single mutants using a threshold for mis-regulation of 2-fold or greater increase or decreasecompared with the wild type, with q , 0.01. We dis-covered that crwn4 mutants displayed the largestnumber of up-regulated loci, with 1,539 genes, com-pared to 455 for crwn1 mutants and 108 for crwn2 mu-tants (Fig. 1A). The number of genes with alteredexpression scaled with the severity of the abnormalnuclei phenotypes in leaf cells. That is, crwn2 plantshave normally shaped nuclei and the least numberof up-regulated genes, while crwn1 mutants exhibitsmaller, round nuclei and a moderate alteration intranscription (Fig. 1A). The most severe misregulationwas seen in the crwn4 mutant (Fig. 1A), which has amore severe effect on nuclear organization, evidencedby a disruption in chromocenter organization and anabnormal nuclear boundary. These results indicatethat loss of a single CRWN protein is enough to elicit

1316 Plant Physiol. Vol. 179, 2019

Choi et al.

www.plantphysiol.orgon October 26, 2020 - Published by Downloaded from Copyright © 2019 American Society of Plant Biologists. All rights reserved.


transcriptomic alterations. The absence of morpho-logical defects on the whole-plant level in crwn singlemutants indicates that the changes in steady-statetranscript levels are tied directly to alterations in nu-clear structure, rather than a result of secondary effectsfrom abnormal growth and development.

Complex Interactions among crwnMutations Are Revealedby Transcriptomic Profiling

The genes misregulated in each crwn mutant formedsets with a high degree of overlap, suggesting sharedfunctions among CRWN proteins. For example,many of the genes that were up-regulated genes inthe crwn1 and crwn2 single mutants were also up-regulated in the crwn4 mutants (Fig. 1A). A similarpattern was observed in the Venn diagrams of down-regulated genes as well as for all misregulated genes(Supplemental Fig. S1).We explored the overlap of function among CRWN

proteins further by analyzing the transcriptomic pro-files of the two crwn double mutants, crwn1 crwn2 andcrwn1 crwn4, which display dwarf phenotypes. Of thetwo, crwn1 crwn2 mutants showed more extensivetranscriptomic misregulation, exceeding the sum of the

transcriptomic profiles of crwn1 and crwn2 single mu-tants (Fig. 1B). Thus, loss of CRWN1 and CRWN2leads to a synergistic effect on transcription (SupplementalFig. S2). This result is consistent with the synergisticphenotypes of crwn1 crwn2 double mutants, whichhave smaller nuclei and suffer from stunted growth,compared with the wild type and crwn1 or crwn2single mutants. The transcriptomic patterns reinforceour previous conclusion that CRWN1 and CRWN2have overlapping functions, but CRWN1, which ismore highly expressed than CRWN2 (;3-fold inwild-type plants in our RNA-seq data set), is suffi-cient to cover for the loss of its close paralog in crwn2mutants.Given the overlap of misregulated genes in crwn1

and crwn4 single mutants, it was surprising to observethat the combination of crwn1 and crwn4 mutationsresulted in a smaller perturbation of gene expressioncompared with crwn4 mutants (Fig. 1C; SupplementalFig. S2). This result was also unexpected because crwn1crwn4 mutants have intermediate whole-plant pheno-types and a nuclear size reduction between that of thesingle mutants and the crwn1 crwn2 double mutant.However, the transcriptomic data demonstrating thatcrwn1 suppresses the crwn4 mutation are consistentwith our previous observations that CRWN1 and

Figure 1. The patterns of up-regulated genes in crwnmutants demonstrate both synergistic and antagonistic relationships amongcrwnmutations. The sizes of each circle in the Venn diagrams are proportional to the number of genes up-regulated at least 2-foldin our RNA-seq data (q, 0.01). A, Up-regulated genes in three crwn single mutants, crwn1, crwn2, and crwn4, showing sharedtranscriptomic alterations. B, Up-regulated genes in the double mutant crwn1 crwn2 and corresponding single mutants, crwn1and crwn2, showing the synergistic effects of the crwn1 and crwn2mutations. C, Up-regulated genes in the doublemutant crwn1crwn4 and corresponding singlemutants, crwn1 and crwn4, showing the antagonistic effects of thesemutations. D, Up-regulatedgenes in two double mutants, crwn1 crwn2 and crwn1 crwn4, and one single mutant, crwn4, illustrating overlapping profiles.Note that PR1 and PR2 fell in the overlap between crwn1 crwn2 and crwn1 crwn4mutants (this overlap contains 119 genes), andPR5 was found only in the sector corresponding to the crwn1 crwn2 mutant (region with 565 genes).

Plant Physiol. Vol. 179, 2019 1317

Loss of CRWN Proteins Induces SA Defense Responses


http://www.plantphysiol.org/cgi/content/full/pp.18.01020/DC1






CRWN4 have opposing effects on chromocenter ag-gregation (Wang et al., 2013).

To summarize, our transcriptomic profiling uncov-ered a complex set of interactions among CRWN genes.On one hand, the gene expression results confirm thatCRWN1 and CRWN2, close paralogs in the same clade,have overlapping functions. In contrast, CRWN1 andCRWN4, which lie in distinct clades, appear to have atleast partially antagonistic functions.

Misregulated Transcripts in crwn Mutants Are EvenlyDistributed on Chromosome Arms But Depleted fromPericentromeric Regions

Next, we determined if genes misregulated in crwnmutants have a nonrandom distribution across the ge-nome. Plant genomes make nonrandom contacts withthe nuclear periphery (Bi et al., 2017), as is the case inanimals, where a subset of the genome is partitionedinto LADs (van Steensel and Belmont, 2017). Therefore,we investigatedwhether or not particular regions of thefolded genome were preferentially affected in crwnmutants. An uneven distribution of misregulated genescould also reflect differential effects on distinct epige-netic compartments in the genome. To investigate thesepossibilities, we drew chromosomal maps marking thepositions of misexpressed transcripts. As shown inSupplemental Figure S3, misregulated loci were evenlydistributed across each chromosome arm but signifi-cantly depleted from pericentromeric regions. The lackof differentially expressed genes in these regions couldindicate that transposable elements (TEs), which areconcentrated in heterochromatic regions flanking thecentromere, are not derepressed in crwn mutants.However, the paucity of TEs detectedmight result froma bias in our RNA-seq analysis based on uniquelymapped reads and a gene-based annotation. Accord-ingly, we used TEtranscripts, an independent programdesigned to analyze differential expression of TEs,allowing transcripts to be mapped to families of ele-ments (Jin et al., 2015). This supplemental analysis de-termined that only a small number of TE familiesshowed modest expression changes (SupplementalTables S1 and S2), and, in most cases, the detected dif-ferences reflected changes in transcript levels fromgenes that contain an embedded TE. For example,transcripts from AT2TE56040, an element in theVANDAL5A subfamily, were ;16-fold higher incrwn1 crwn2 mutants compared with the wild type(Supplemental Table S1), with no significant over-expression in crwn1 or crwn2. However, inspection ofthe RNA-seq reads indicated that this apparent over-expression was due to longer transcripts correspondingto AT2G30020, which contains the element within anexon. Taken as a whole, our results indicate that epi-genetic silencing of TEs is not significantly reduced inany of the crwn mutants, despite the significant dis-persion of chromocenters observed in crwn4 nuclei(Wang et al., 2013).

We then characterized the chromatin states of mis-regulated genes in crwnmutants to determine if certainepigenetic signatures were overrepresented or under-represented. For this analysis, we used the nine chro-matin categories established by Sequeira-Mendes et al.(2014) to define the landscape of the Arabidopsis epi-genome. We found that up-regulated genes in crwnmutants showed a higher proportion of genes in chro-matin state 2 compared with either the whole Arabi-dopsis gene list or the genes that were tested fordifferential expression (Supplemental Fig. S4). Chro-matin state 2 is characterized by active marks (e.g.histone H3K4me2 and H3K4me3) associated withtranscription as well as a high level of the repressivenucleosome modification, histone H3K27me3. Theoverrepresentation of chromatin state 2 coincided withan underrepresentation of states 1 and 3, which areassociated with actively expressed genes and tran-scriptional elongation, respectively. The shift in repre-sentation of chromatin states among up-regulatedtargets suggests that CRWNs might contribute to themodulation of H3K27me-associated chromatin or dif-ferentially affect inducible genes that are packaged inchromatin state 2.

Analysis of Gene Ontology of Misregulated Genes ShowsEctopic Defense Responses in crwn Mutants

Next, we asked what kind of genes are misregulatedin the RNA-seq profiles of crwnmutants. We found twopatterns in this analysis. First, up-regulated genes werecharacterized by stress-associated Gene Ontology (GO)terms (Table 1). Althoughwe found that both biotic andabiotic stress-related GO terms were identified, we fo-cused on biotic stress because the GO terms with thehighest fold enrichment were those related to plantdefense (e.g. response to chitin and response to mole-cule of bacterial origins). The second pattern was anenrichment of genes associated with metabolism andother stress pathways among the down-regulated loci(Supplemental Table S3). Examples of these down-regulated pathways include glucosinolate biosynthe-sis, wax biosynthesis, and cell wall biogenesis.Together, these two observations suggest that a loss ofCRWN1 or CRWN4, or a combination of CRWN paral-ogs, leads to an induction of defense-related genes anda down-regulation of a subset of metabolic processes.

Two crwn Double Mutants Exhibit DC3000 Resistance andElevated PR Gene Expression

Our analysis of transcriptomic data uncovered anectopic induction of defense responses in crwnmutants,prompting us to test if there are any defense pheno-types in these mutants. We infected an array of crwnmutants with two commonly studied Arabidopsispathogens representing both fungi and bacteria: thenecrotrophic fungal pathogen Botrytis cinerea or the


Choi et al.









Table 1. Statistical overrepresentation test for up-regulated genes in crwn mutants

InputGO Terms (Biological Process

Complete)

Total No. of

Arabidopsis Genes

in the Term

Input

Genes No.

Expected

No. of GenesFold Enrichment P

Up-regulated genes incrwn1 mutants (444genes mapped)

Response (rsp.) to chitin 108 19 1.76 10.76 1.19E-10Rsp. to jasmonic acid 174 18 2.84 6.33 2.68E-06Rsp. to wounding 179 17 2.93 5.81 2.66E-05Ethylene-activated signaling

pathway135 11 2.21 4.99 4.08E-02

Rsp. to SA 164 13 2.68 4.85 9.72E-03Rsp. to abscisic acid 413 25 6.75 3.70 8.24E-05Rsp. to salt stress 417 24 6.81 3.52 3.82E-04Protein phosphorylation 828 47 13.53 3.47 6.30E-10Rsp. to bacteria (bac.) 353 20 5.77 3.47 5.12E-03Cellular rsp. to acid chemical 346 19 5.65 3.36 1.41E-02

Up-regulated genes incrwn2 mutants (107genes mapped)

Rsp. to organonitrogen compound 133 6 0.52 11.53 3.42E-02Rsp. to cold 281 9 1.1 8.19 4.02E-03Rsp. to salt stress 417 10 1.63 6.13 1.35E-02Rsp. to acid chemical 896 21 3.51 5.99 9.39E-08Rsp. to oxygen-containing

compound1,156 22 4.52 4.86 1.51E-06

Rsp. to inorganic substance 690 12 2.7 4.45 3.88E-02Rsp. to hormone 1,218 18 4.76 3.78 2.65E-03

Up-regulated genes incrwn4 mutants(1,510 genes mapped)

Rsp. to chitin 108 57 5.98 9.53 1.22E-32Rsp. to molecule of bac. origin 20 8 1.11 7.22 4.54E-02Defense rsp. to fungus,

incompatibleinteraction

37 14 2.05 6.83 8.20E-05

Defense rsp. to bac., incompatibleinteraction

40 11 2.22 4.96 4.48E-02

Rsp. to wounding 179 46 9.91 4.64 1.09E-13Plant-type hypersensitive rsp. 67 17 3.71 4.58 8.54E-04Rsp. to SA 164 39 9.08 4.29 2.75E-10Protein autophosphorylation 115 27 6.37 4.24 1.98E-06Cellular rsp. to jasmonic acid

stimulus61 14 3.38 4.14 2.67E-02

Ethylene-activated signalingpathway

135 29 7.48 3.88 3.47E-06

Up-regulated genes incrwn1 crwn2 mutants(1,686 genes mapped)

Rsp. to chitin 108 62 6.68 9.27 5.61E-35Rsp. to molecule of bac. origin 20 9 1.24 7.27 1.33E-02SA-mediated signaling pathway 35 14 2.17 6.46 1.61E-04Defense rsp. to bac., incompatible

interaction40 15 2.48 6.06 1.27E-04

Plant-type hypersensitive rsp. 67 24 4.15 5.79 3.97E-08Systemic acquired resistance 44 15 2.72 5.51 4.24E-04Regulation of cell death 58 16 3.59 4.46 2.64E-03Regulation of rsp. to biotic stimulus 49 13 3.03 4.29 3.82E-02Regulation of rsp. to external

stimulus53 14 3.28 4.27 1.94E-02

Positive regulation of defense rsp. 64 16 3.96 4.04 9.07E-03Up-regulated genes in

crwn1 crwn4 mutants(382 genes mapped)

Toxin metabolic process 54 8 0.76 10.53 2.89E-03Cellular rsp. to SA stimulus 50 7 0.7 9.95 1.91E-02Defense rsp. to bac. 281 29 3.96 7.33 5.01E-13Rsp. to organonitrogen compound 133 13 1.87 6.94 1.85E-04Innate immune rsp. 239 16 3.36 4.76 9.87E-04Secondary metabolite biosynthetic

process256 14 3.6 3.89 4.85E-02

Rsp. to abscisic acid 413 22 5.81 3.78 3.62E-04Rsp. to fungus 490 26 6.9 3.77 2.90E-05Rsp. to oxidative stress 364 17 5.12 3.32 4.89E-02Protein phosphorylation 828 29 11.65 2.49 1.97E-02





hemibiotrophic bacterial pathogen P. syringae (DC3000).We did not detect differences among the mutants chal-lengedwith B. cinerea; however, we found that the crwn1crwn2 mutant significantly suppressed DC3000 growthin leaf tissue, compared with the wild type and singlecrwn mutants (Fig. 2A), while the crwn1 crwn4 mutantexhibited an intermediate phenotype.

These graded disease phenotypes paralleled the ex-pression of Pathogenesis Related (PR) marker genes inuninfected crwn mutants. Reverse transcription quan-titative PCR (RT-qPCR) assays showed that PR1, PR2,and PR5 were highly expressed in both crwn1 crwn2and crwn1 crwn4 double mutants (Fig. 2B), but to amore extreme degree in crwn1 crwn2 mutants. Fur-thermore, despite showing widespread transcriptionalchanges, single crwn1 and crwn4mutants did not showsignificant up-regulation of PR genes, nor did thesegenotypes suppress the growth of DC3000. These RT-qPCR results were consistent with our RNA-seq data,which indicated that PR genes are up-regulated in crwn1crwn2 and crwn1 crwn4 plants but not in crwn singlemutants (Fig. 1D). Thus, the PR gene expression patternindicates that crwn single mutants fail to induce defenseresponses fully, while the crwn1 crwn2 and crwn1 crwn4double mutants induce defense responses above athreshold necessary to interfere with pathogen growth.

crwn Double Mutants Exhibit Cell Death Lesions

Spontaneous defense responses, such as those ob-served in crwn doublemutants, are also characteristic oflesion-mimic mutants (LMMs), which develop ectopiccell death foci similar to hypersensitive response lesions(Moeder and Yoshioka, 2008). As LMMs trigger pre-mature and prolonged defense responses, they alsoshowmany growth phenotypes, such as slower growth

and dwarfism (Bowling et al., 1997; Bruggeman et al.,2015). We assayed cell death in the crwn mutants be-cause crwn double mutants display both autonomousdefense responses and dwarfism. We found that bothcrwn1 crwn2 and crwn1 crwn4 doublemutants exhibiteddead cells in rosette leaves stained with Trypan Blue(Fig. 3A). Consistent with the expression level of the PRgenes and the resistance against DC3000, crwn1 crwn2mutants showed a higher incidence of cell death com-pared with crwn1 crwn4 plants. Almost no cell deathwas found in matched tissues from either single crwnmutants or wild-type control samples.

We checked if spontaneous defense phenotypes in-tensify over time, because young seedlings of crwn1crwn2 mutants have less wrinkled leaves than olderplants. We found no cell death in 12-d-old crwn1 crwn2mutants but lesions were frequent in 22-d-old crwn1crwn2 leaves (Fig. 3A). Moreover, cell death was morewidespread in 30-d-old crwn1 crwn2 plants (Fig. 3A). Asimilar age-dependent increase in cell death was ob-served in crwn1 crwn4 mutants.

Paralleling the increased incidence of cell death, PR1expression was relatively low in leaves from younger14- and 21-d-old crwn1 crwn2 plants but increaseddramatically in 27-d-old plants (Fig. 3B). These resultsshow that perturbation of the nuclear periphery in plantcells leads to age-dependent effects on the whole-plantlevel, manifested here in the form of cell death and geneexpression changes.

crwn1 crwn2 and crwn1 crwn4 Double Mutants Show HighLevels of Total SA

Next, we sought to understand the connectionamong the diverse responses displayed by the crwndouble mutants. SA is a prominent regulator of plantdefense responses, including the hypersensitive

Figure 2. crwn1 crwn2 and crwn1 crwn4mutants exhibit spontaneous defense responses. A, DC3000 growth in rosette leaves of 22-d-old plants. The twodoublemutants have elevated resistance againstDC3000. Log10 values of colony-forming units (cfu) fromeachmutantwere calculated 2 and 4 d after infection (DAI). Error bars indicate SD (n = 4). Each mutant was compared with the wild type (WT) usingStudent’s t test (**, P , 0.01 and ***, P, 0.001). B, RT-qPCR data showing expression of three PR genes in 27-d-old plants. Transcriptlevels of the marker genes are significantly higher in the two double mutants, consistent with their DC3000 resistance phenotype. Errorbars indicate SE (n = 3). Student’s t tests were performed based on delta-Ct values (*, P, 0.05; **, P, 0.01; and ***, P, 0.001).


Choi et al.



response, systemic acquired resistance, and reactiveoxygen species production (Vlot et al., 2009). Conse-quently, we investigated the causal connection betweenthe phenotypes of crwn mutants and SA. First, wemeasured SA abundance in crwn mutants using massspectrometry coupled with HPLC (HPLC/MS). SA canexist as several derivatives, including SA glucoside andsalicylate glucose ester. The glucosylation of SA isknown to reduce its toxicity and allows storage of largequantities of the hormone (Dempsey et al., 2011).The level of free SA was more than 2-fold higher incrwn1 crwn2 mutants compared with the wild type orcrwn1, crwn2, and crwn4 single mutants (Fig. 4A). Forglucosylated SA, the fold change wasmuch higher thanthat for free SA: we recorded a 14-fold increase ofglucosylated SA in crwn1 crwn2 mutants and a morethan 6-fold increase in crwn1 crwn4 mutants (Fig. 4B).

Thus, we found that both free SA and glucosylated SAare elevated in crwn1 crwn2 mutants, while onlyglucosylated SA is highly abundant in crwn1 crwn4mutants. These results are consistent with the inter-mediate defense phenotypes in crwn1 crwn4 plantscompared with crwn1 crwn2 mutants and the lack ofsignificant defense phenotypes in crwn single mutants.These findings suggest that SA is an important media-tor of the abnormal gene expression and defenseresponses exhibited by crwn mutants.

SA Is Responsible for a Significant Portion ofMisregulated Genes in crwn1 crwn2 Mutants

To test the prediction that SA overaccumulation isresponsible for the altered transcriptome in crwn

Figure 3. crwn double mutants exhibit cell death and PR gene expression, which are exacerbated with aging. A, TrypanBlue staining showed that crwn1 crwn2 and crwn1 crwn4mutants form spontaneous lesions. As plants matured, cell deathwas more apparent and more frequent. B, PR1 expression in leaves from crwn1 crwn2mutants of different ages. Expressionof PR1 is higher in chronologically older leaves of crwn1 crwn2 mutants, consistent with our observation that older plantsdevelop more lesions. PR1 expression at each stage was normalized to transcript levels in wild-type samples for thehousekeeping gene ROC1. Error bars indicate SE (n = 3). Student’s t tests were performed based on delta-Ct values (*, P ,0.05 and ***, P , 0.001). C, The sid2-1 mutation suppressed cell death in 22-d-old crwn1 crwn2 mutants; however, 30-d-old plants still exhibited some lesion formation. Bars = 100 mm.





mutants, we compared our RNA-seq data of crwn1crwn2 mutants with a microarray data set from SA-treated wild-type plants (Zhou et al., 2015). About40% of the up-regulated genes in the SA-treated wild-type sample overlapped with the up-regulation profileof crwn1 crwn2 mutants (Fig. 4C). GO term analysisrevealed that genes in the overlap of these sets areclosely related to bacterial resistance and SA response(Supplemental Table S4). Moreover, 25% of the down-regulated genes in the SA-treated wild-type sampleoverlapped with those down-regulated in crwn1 crwn2samples (Fig. 4C). Among the genes down-regulated inboth data sets are those featuring the response to jas-monic acid GO term (Supplemental Table S4), reflectingthe well-known antagonism between SA and jasmonicacid signaling (Robert-Seilaniantz et al., 2011).

The number of loci misregulated in both SA-treatedplants and crwn single mutants was reduced relative

to the overlap between SA-treated and crwn1 crwn2double mutant samples (Supplemental Fig. S5). Thispattern is consistent with the fact that the crwn singlemutants do not accumulate high levels of SA. However,this explanation is incomplete, as the overlap is rela-tively modest between the SA-treatment data set andthe misregulated loci in crwn1 crwn4 mutants withelevated SA levels. Taken together, our findings indi-cate that SA signaling accounts for a significant portionof the transcriptomic changes in the crwn1 crwn2 mu-tants but that crwnmutations cause gene misregulationthrough additional mechanisms.

SID2 Transcripts Are Abundant in Two crwnDouble Mutants

To understand the mechanism responsible for SAaccumulation in crwn1 crwn2 and crwn1 crwn4mutants,

Figure 4. SA levels in crwn1 crwn2 and crwn1 crwn4mutants are significantly higher than in wild-type (WT) plants or crwn singlemutants. A, HPLC/MS detection of free SA levels from 27-d-old plants. Peak intensities of free SA per fresh sample weight werenormalized to the spiked internal control d4-SA. Each mutant was compared with the wild type using Student’s t test, and error barsindicate SD (*, P, 0.05; n=3). B,HPLC/MSdetection of glucosylated SA (SA-Glc, indicating either SA glucoside or salicylateGlc ester)levels from each mutant. The detection method, units, and statistical analysis were identical to those described for A (*, P, 0.05 and**, P, 0.01). C, Overlap of misregulated genes in our RNA-seq data from crwn1 crwn2mutants and those in microarray data from1 mM SA-treated wild-type plants (Zhou et al., 2015). For SA-treated wild-type microarray data, genes at least 2-fold increased ordecreased with adjusted P, 0.05 were used. For the RNA-seq of crwn1 crwn2mutants, the criteria described in Figure 1 were used.The significance of the overlap of both up-regulated and down-regulated gene comparisons was assessed by the hypergeometric test;the resulting P values in both cases were far less than 0.001. D, RT-qPCR data showing expression of the SID2 gene in 27-d-old plants.Error bars indicate SE (n = 3). Student’s t tests were performed based on delta-Ct values (*, P , 0.05 and **, P , 0.01).


Choi et al.






we checked for changes in the transcript levels of genesinvolved in SA biosynthesis. There are two differentSA biosynthetic pathways in plants (Rivas-SanVicente and Plasencia, 2011). First, the phenyl-propanoid pathway utilizes Phe in the cytoplasmto produce SA. Phenylalanine ammonia lyases(PALs) are important enzymes in this pathway. Thesecond pathway begins with isochorismate in thechloroplast. In the latter pathway, ICS, where ICS1is a synonym of SID2, are key enzymes responsiblefor SA biosynthesis (Seyfferth and Tsuda, 2014).SID2 is highly expressed when plants are infectedwith P. syringae pv maculicola (Wildermuth et al.,2001). We found that crwn1 crwn2 mutants expressvery high levels of SID2 transcripts, while tran-scription of PAL1 and PAL4 is reduced by morethan half in our RNA-seq data (Supplemental Fig.S6). Using RT-qPCR, we validated that the SID2transcript level was significantly increased incrwn1 crwn2 mutants, while crwn1 crwn4 mutantsexhibited an intermediate level of expression(Fig. 4D). These results are consistent with the totalSA levels and the magnitude of defense responsesin the crwn mutants. In addition, we found thatthe genes encoding several positive regulatorsof SID2 (Seyfferth and Tsuda, 2014), includingSARD1 and CBP60g, are highly up-regulated in thetranscriptomic profiles of crwn1 crwn2 mutants(Supplemental Fig. S6). These results indicate thatloss of CRWN genes leads to ectopic pathogen sig-naling by ramping up the production of SA viaoverexpression of SID2.

Blocking Induced SA Biosynthesis Diminishes But DoesNot Abolish Lesion Formation

After we observed that abnormally high SA levelswere synthesized in crwn double mutants, we intro-duced a sid2-1 mutation to block SA biosynthesisto determine which crwn phenotypes are depen-dent on SA. As shown in Supplemental Figure S7, thesid2-1 mutation effectively suppressed SA accumu-lation in the crwn1 crwn2 background. The lack ofSA is consistent with the fact that DC3000 growthin sid2 crwn1 crwn2 mutants was comparable to thatin sid2 mutants (Supplemental Fig. S8). Therefore,the sid2-1 allele was epistatic to the crwn mutationsfor both SA accumulation and bacterial growthphenotypes, indicating that the primary reason forectopic defense responses in crwn mutants is a highlevel of SA.We observed that the sid2-1 mutation also sup-

pressed lesion formation in crwn1 crwn2 mutants, but,in this case, the suppression was incomplete. While nocell death was observed in 22-d-old sid2 crwn1 crwn2leaves (Fig. 3C), scattered lesions were still evident in30-d-old leaf tissue. This result demonstrates that somecell death occurs in crwn1 crwn2 mutants in an SA-independent manner.

crwn1 crwn2 Mutants Exhibit SA-IndependentDevelopmental Defects

Anumber of additional phenotypes exhibited by crwn1crwn2 mutants are independent of SA accumulation tovarying degrees. For example, the wrinkled leaves anddwarf stature of crwn double mutants were only par-tially suppressed by disruption of SID2 (SupplementalFig. S9). In particular, sid2 crwn1 crwn2 plants stillexhibited some dwarfism, most noticeably in boltstature (Supplemental Fig. S9C). These results indi-cate that a large component of the dwarfing syndromedisplayed by crwn1 crwn2 mutants is caused by SAoveraccumulation, although there is still an SA-independent pathway affecting bolt stature.Seed shape was also altered in certain crwn mutants,

especially crwn1 crwn2. As shown in SupplementalFigure S10, crwn1 crwn2 seeds often had deeplycreviced surfaces, and we observed a range of seedmorphologies, from normal to smaller and darkerseeds. Addition of the sid2-1 allele did not have anappreciable effect on these phenotypes, as seedsharvested from sid2 crwn1 crwn2 mutants were alsomisshapened.We also examined nuclei in the sid2 crwn1 crwn2

mutant to determine if the reduced organellar size andabnormal spherical shape characteristic of this geno-type might be partially attributable to ectopic SAsignaling. As shown in Supplemental Figure S11,elongated anther filament cells of sid2 crwn1 crwn2mutants retained the small, round nuclear morphologyobserved in crwn1 crwn2 mutants. Therefore, the sid2-1mutation had no effect on nuclear size or shape. Thisfinding demonstrates that the nuclear phenotypescaused by loss of CRWN1 and CRWN2 are primaryeffects, not downstream consequences of elevatedSA production in these mutants.

DISCUSSION

To investigate the importance of three-dimensionalgenome organization within the nucleus, we asked ifperturbation of the plant nuclear periphery leads toaberrant gene expression patterns. We found that de-ficiencies of individual NMCP proteins in Arabidopsis,particularly CRWN1 or CRWN4, led to changes in thesteady-state levels of a large number of transcripts, andeven more transcripts were altered in crwn1 crwn2double mutants. A large portion of the overexpressedgenes were involved in pathogen defense. The proxi-mate cause of much of this misregulation is the over-production of the defense signaling molecule SA as aconsequence of the up-regulation of the SA biosyntheticgene, SID2. A constellation of phenotypes, includingelevated bacterial resistance, dwarfism, cell death le-sions, and wrinkled leaves, could be either fully orpartially suppressed by the null sid2-1 mutation. Usingthis approach, we also identified consequences of nu-clear defects that are independent of SA signaling, such
















as reduced plant stature, abnormal seed shape, and abaseline level of cell death.

Synthesizing our findings, we propose two models(Fig. 5) to explain this complex response to alteringnuclear structure in plants. The initiating event in bothscenarios is the nuclear defect caused by genetic abla-tion of a subset of CRWNnuclear periphery proteins. Inthe firstmodel (Fig. 5A), these defects lead to expressionchanges in vanguard genes, including SID2, whichleads to SA production and secondary changes tothe transcriptome as well as downstream effects onpathogen defense and plant morphology. Severalmechanisms might be operating to disrupt gene ex-pression, such as the direct involvement of CRWNproteins in transcriptional regulation (either as part ofan NL or as free proteins; see below) or changesin three-dimensional positioning of genes. Supportfor the latter mechanism was recently reported formouse embryonic stem cells, where changes inthree-dimensional genome structure due to lamindeficiency led to altered gene expression patternswithin LADs (Zheng et al., 2018). Furthermore, anincrease in trans- versus cis-chromosomal contactswas detected via Hi-C analysis of crwn1 and crwn4mutants, suggesting that NL changes could reducelocal looping interactions or alter other three-dimensional configurations important for tran-scriptional regulation in interphase plant nuclei(Grob et al., 2014).

More localized changes in chromatin could also playa role, mirroring the direct effects that the nuclear pe-riphery exerts on histone modification or chromatin-mediated silencing in animals and fungi (Harr et al.,2015, 2016). Recent work from Schubert and col-leagues in Arabidopsis demonstrated a physical andfunctional connection between CRWN1 and PWWPINTERACTORS OF POLYCOMBS1, a protein associ-ated with the Polycomb Repressive Complex 2 re-sponsible for H3K27methylation (Mikulski et al., 2017).This mechanism is consistent with our observation thatloci packaged in chromatin state 2, featuring both active

and repressive (e.g. histone H3K27me3) epigeneticmarks, are overrepresented among up-regulated genesin crwn mutants (Supplemental Fig. S4). This result,however, could also reflect the fact that SA-modulatedgenes tend to be packaged in chromatin state 2; ap-proximately 40% of genes overexpressed in response toSA contained at least one domain of state 2 chromatin(relative to the expected occurrence of approximately25%; Supplemental Fig. S12). We investigated the pos-sible role of H3K27me3 regulation via chromatin im-munoprecipitation (ChIP)/qPCR experiments in differentcrwngenotypes, using the SA-regulated locusPR1, aswellas AGAMOUS (AG) as a control locus. AG is a well-known target of H3K27me3 repression that is notknown to be regulated by SA (Zhang et al., 2007). Asshown in Supplemental Figure S13, we observed a trendtoward reducedH3K27me3modification at the PR1 locusin crwn1 crwn2mutants (below the cutoff for significance)but no change in crwn1 mutants. These results are con-sistent with the increase in PR1 expression in the doublebut not the single mutant. These findings could beinterpreted as evidence that histone H3K27me3changes, mediated by the loss of CRWN proteins,play a role in PR1 gene induction, but it is possiblethat chromatin modification changes lie downstreamof SA-modulated transcription. It is important to notethat the ultimate targets of misregulation in crwnmutants are genes in the chromosome arms, rather thanthe heterochromatic regions surrounding the centro-mere or TEs (Fransz et al., 2002). Our results aresupported by previous findings from Poulet et al.(2017) showing that crwn1 crwn2 mutants do notinduce transcription of transposons, such as TSI, orcentromeric repeats.

The second model (Fig. 5B) does not invoke a role forCRWN proteins or nuclear organization in transcrip-tional control; rather, it posits that the cascade of tran-scriptional changes lies downstream of cell death orcell damage perception caused by nuclear defects(e.g. leakage [Denais et al., 2016] or abnormal connec-tion to the cytoskeleton). This model explains the

Figure 5. Two models to explain how crwn mu-tations affect disease signaling and other pheno-types. A, The first model posits that crwnmutationslead to transcriptional changes via the disruption inthe direct action of CRWN proteins or epigeneticmechanisms (e.g. through changes in chromatinmodification or three-dimensional genomic con-figuration). In this first model, overexpression ofSID2 and subsequent SA accumulation lead topathogen defense phenotypes and an LMM syn-drome. B, In the alternative model, cell damageand/or death due to nuclear dysfunction is theprimary effect of crwn mutations. Downstreameffects of age-dependent cell damage/death areamplified via an SA-mediated pathway, but anSA-independent pathway also mediates devel-opmental defects.


Choi et al.






age-dependent effects seen in crwn mutants, in whichgene expression changes and the incidence of cell deathlesions progress in their intensity over time, as a con-sequence of accumulated cell damage and death. In thesecond model, SA production lies downstream of thisdamage and cell death and leads to an amplificationand proliferation of cell death in a feed-forward man-ner. Key support for this model is the low incidence ofcell death in the absence of induced SA production inthe sid2 crwn1 crwn2 mutant (Fig. 3C). This result sug-gests that age-dependent cell death/damage precipitatedbynuclear changes initiates the subsequent SA-dependentlesion-mimic syndrome, with its characteristic dwarfing,wrinkled leaves, and PR gene induction. The age-dependent effects seen in crwn mutants are consis-tent with the types of progressive changes in nuclearstructure that are associated with both natural agingas well as progeria syndromes caused by humanlaminopathies (Scaffidi and Misteli, 2006).Several recent publications shed light on these

models and highlight the importance of the plant nu-cleus in defense signaling. Most relevant is a recentlypublished study claiming that CRWN1 acts as a core-pressor, with NAC WITH TRANSMEMBRANE MO-TIF1-LIKE9 (NTL9), of PR genes (Guo et al., 2017).These data support aspects of the transcriptional con-trolmodel (Fig. 5A) inwhich CRWNproteins can play adirect role in transcriptional regulation. However, thisspecific corepressor activity for CRWN1 with NTL9fails to explain the up-regulation of SID2, as NTL9 is anactivator of this SA biosynthetic locus (Zheng et al.,2015). An alternative possibility is that CRWN1 inter-acts with NTL9 to block its activation of SID2 by se-questering the transcription factor at the nuclearperiphery. We note that Guo et al. (2017) did notobserve a spike in SA production in crwn1 crwn2mutants, which we demonstrate here (Fig. 4), an ob-servation that can account for PR gene induction aswell as the broader changes in gene expression andthe elevated bacterial pathogen resistance exhibitedby these mutants. The increase in SA levels in crwndouble mutants can also explain the observed in-duction of reactive oxygen species recently noted byWang et al. (2019), which they associated with theirindependent observation of the elevated level ofcell death.The role of the nuclear structure in defense signaling

has also been brought into focus by studies showing thatplant nucleoporins play important roles. For example,Gu et al. (2016) demonstrated that CONSTITUTIVEEXPRESSION OF PR GENES5 (CPR5; Bowling et al.,1997), long known for its roles in systemic acquiredresistance, is a nucleoporin that regulates the release ofcyclin-dependent kinase inhibitors and the transport ofsignaling complexes. Furthermore, they showed thatcpr5mutants, which are LMMs exhibiting spontaneousdefense response, have round nuclei similar to those ofcrwn mutants. These findings suggest that the nuclearstructural changes that occur in cpr5 and crwn mutantsmight be mechanistically related. For instance, defects

at the nuclear periphery could alter the organization oractivity of nuclear pore complexes (Al-Haboubi et al.,2011; Guo et al., 2014). Other recent work points tonuclear pores as a site of defense signaling in plants.Tamura et al. (2017) reported that the nucleoporinmutant nup136 shows compromised resistanceagainst DC3000, while Gu et al. (2016) suggestedthat the same mutants have enhanced effector-triggered immunity resistance against another bac-terial pathogen (Psm/AvrRpt2). Although these twostudies suggest different roles of this particularnucleoporin, it is now indisputable that componentsat the nuclear periphery participate in regulation ofplant defense responses.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants were grown onMetroMix 360 soil(Sun Gro Horticulture) under long-day conditions (16 h of light/8 h of dark)at approximately 22°C in environmental growth chambers after 2 d of strat-ification in a 4°C cold room. All genotypes are in the Columbia background.The crwn mutant material used was described by Wang et al. (2013); theoriginal allele sources are crwn1 (SALK_025347), crwn2 (SALK_076653), andcrwn4 (SALK_079296RP), which were acquired from the Arabidopsis Bio-logical Resource Center at Ohio State University. The sid2-1 mutant materialoriginated from the Metraux group (Nawrath and Métraux, 1999). The sid2-1 crwn1 crwn2 triple mutants were generated by crossing sid2-1 (♀) mutants tocrwn1 crwn2 (♂) mutants and subsequent segregation. The genotypes of allthe mutants used in this study were identified by PCR amplification withprimers listed below and reference to marker polymorphisms (SupplementalTable S5).

RNA-Seq and Bioinformatic Analysis

Three biological replicates from each genotype were used for RNA-seq(National Center for Biotechnology Information [NCBI] SRA BioProject IDPRJNA485018). Rosette leaves from 4-week-old adult plants were used toextract total RNA using TRIzol reagent (Thermo Fisher Scientific) and fur-ther purified with the Qiagen RNeasy kit. RNA samples were processed bythe Genomic Core Facility at Cornell’s Weill Medical School using a stan-dard Illumina protocol to construct single-stranded mRNA libraries. TheIllumina Hi-Seq 2000 platform was used for the sequencing. The resulting 51nucleotide reads were analyzed using the Tuxedo Suite software (Trapnellet al., 2012). Briefly, Bowtie2 (v. 2.2.2) and Tophat2 (v. 2.1.1) were used tobuild an index for the TAIR10 Arabidopsis genome to map RNA-seq readsand to identify splice junctions between exons (Arabidopsis GenomeInitiative, 2000; Langmead et al., 2009; Kim et al., 2013). Finally, Cufflinks(v. 2.2.1) was used to calculate differential gene expression (Trapnell et al.,2010). GO term analysis was conducted using PANTHER (Mi et al., 2017).The overrepresentation test (released July 15, 2016) was performed withmisregulated gene sets against the annotation data set GO biological processcomplete (GO ontology database released November 30, 2016). Note that thenumbers of genes listed for the different GO terms in Table 1 do not matchthe values in the Venn diagrams in Figure 1 and Supplemental Figure S1because not all genes in our data set are assigned GO terms in the PANTHERdatabase.

Venn diagrams for all five genotypes in Supplemental Figure S1 weregenerated using InteractiVenn (Heberle et al., 2015).

For TE analysis, STAR (v. 2.5.3a_modified) was used for index building andread mapping (Dobin et al., 2013). Mapping employed -winAnchorMulti-mapNmax 100 and -outFilterMultimapNmax 100 options. Next, TEtran-scripts (v. 1.5.1) was used to analyze TE expression in alignment files fromthe STAR output (Jin et al., 2015). The annotation GFF3 file for genes wasobtained from TAIR10 and converted to GTF by gffread (Trapnell et al.,2010) with -FTo option. The TE annotation file was provided by theHammell lab (http://labshare.cshl.edu/shares/mhammelllab/www-data/








http://labshare.cshl.edu/shares/mhammelllab/www-data/TEToolkit/TE_GTF/


TEToolkit/TE_GTF/). The GTF file from the Hammell lab grouped TEs byfamily name. We also modified this file so that reads could be mapped toindividual TEs. Read count tables produced by TEtranscripts were analyzedby DESeq2 (Love et al., 2014).

For the comparison between SA-treated wild type and crwn mutants,microarray data from 23-d-old wild-type plants sampled 3 h after 1 mM SAtreatment (GSM1496067, 1496075, and 1496083) and water-treated controls(GSM1496065, 1496073, and 1496081) were obtained from the NCBI GeneExpression Omnibus database (series no. GSE61059; Zhou et al., 2015).Genes misregulated more than 2-fold with adjusted P , 0.05 werescreened with GEO2R using default options (Edgar et al., 2002; Barrettet al., 2013). The resulting list of misregulated microarray elements wereconverted to locus identifiers by TAIR Microarray Elements Search andDownload tool. These genes were compared with our RNA-seq data fromthe crwn mutants.

Our chromatin state analysis was based on the study by Sequeira-Mendeset al. (2014) of the epigenetic landscape of the wild-type Arabidopsis ge-nome. We overlapped chromatin states of Arabidopsis genomes onto mis-regulated genes in crwn mutants by using Intersect the intervals of two datasets under the Operate on Genomic Intervals tool in Galaxy (Afgan et al.,2016). Minimum overlapping was set as 200 bp, which is the length of anucleosome. Because of this overlapping definition, one gene can exist asmore than two states.

To examine the distribution of misregulated genes across the genome,chromosomal maps were drawn based on the Columbia reference genomesequence (TAIR10) with the genoPlotR package (Guy et al., 2010). Pericentro-meric regions were marked based on Stroud et al. (2013). Genes depicted on themap are the same as the ones used to draw the Venn diagrams in Figure 1 andSupplemental Figure S1.

Pseudomonas syringae DC3000 Bacterial Growth Assays

DC3000 was grown on King’s B medium (20 g L21 Proteose Peptone no. 3,10 mL L21 glycerol, 0.4 g L21 MgSO4, 1.5 g L21 K2HPO4, and 18 g L21 agar)containing 50 mg mL21 rifampicin and 50 mg mL21 kanamycin. Bacterial cellswere collected and suspended in 10 mM MgCl2 solution to a final concen-tration of 105 cells mL21. The abaxial sides of three fully expanded rosetteleaves of 22-d-old plants were infiltrated with the bacterial suspension usinga syringe without a needle. Four plants per genotype were used for biologicalreplicates. Infected leaves were harvested 2 and 4 d after inoculation. For theset of infections investigating the effect of sid2mutations, only 0 and 2 d afterinoculation were checked. Bacterial growth experiments were repeatedmultiple times, with each replicate showing a significant reduction of DC3000growth in the crwn double mutants.

RNA Extraction and RT-qPCR

Aboveground shoot material, excluding bolts and flowers, was harvestedand frozen in liquid nitrogen. Frozen samples were ground using three metalbeads or mortars and pestles. The E.Z.N.A. Plant RNAKit (Omega Bio-tek) wasused to extract total RNA, followed by DNase I (New England Biolabs) treat-ment. cDNA synthesis was performed with either the SuperScript III or Su-perScript IV First-Strand Synthesis System (Thermo Fisher Scientific) usingoligo(dT) primers. For the RT-qPCR, iTaq Universal SYBR Green Supermix(Bio-Rad) was used. Three biological replicates were tested (plant cohortsgrown independently at separate times; one plant per sample), and eachreplicate consisted of at least three reactions per target gene. The size ofamplicons was designed to be approximately 100 bp. A CFX 96 Real-TimeSystem (Bio-Rad) mounted on a C1000 Thermal Cycler (Bio-Rad) was used todetect products. The results were analyzed using the Bio-Rad CFX Manager3.1. software package.

Cell Death Lesion Detection and Imaging

Fully expanded rosette leaves were used for cell death lesion analysis.Whenever possible, the fifth leaf that emerged from the meristem was used.However, similar patternswere found inall fully expanded leaves of similar size.Leaveswere harvested and stained as described (vanWees, 2008). Briefly, leaveswere immersed in 2.5 mg mL21 Trypan Blue-lactophenol solution with 2 vol-umes of ethanol added. Samples were then boiled for 1 min and left on a shakerat room temperature for 2 to 3 h. Destaining was performed twice with 2.5 g

mL21 chloral hydrate solution. Final samples were stored with 70% (v/v)glycerol and observed with a Leica DM5500B microscope with 103 magnifi-cation to check stained foci.

SA Extraction and HPLC/MS Analysis

Extraction of SAwas performed as indicated in the protocol from Stingl et al.(2013) with slight modifications. Briefly, leaves were frozen in liquid nitro-gen and ground using metal beads. Samples were suspended with 950 mL ofethylacetate:formic acid solution (99:1, v/v). Tomake the d4-SA internal controlsolution, d6-SA (#616796; Sigma-Aldrich) was dissolved in methanol. Fiftymicroliters of 7.5 ng mL21 d4-SA solution was added to the sample as an in-ternal control. Nine hundred microliters of the supernatants was isolated, andsolvents were evaporated with a SpeedVac concentrator (Savant ISS110;Thermo Scientific). The final residues were dissolved in 100 mL of acetonitrile:water (1:1, v/v) solution and filtered with a 0.45-mmMultiScreenHTSHV FilterPlate (MSHVN4510; Millipore).

The analytical reverse-phase separation was performed with a Dionex Ulti-mate 3000 Series LC systemwith a Titan C18 UHPLC Column (7.5 cm3 2.1 mm,1.9 mm; Sigma-Aldrich) at 40°C. The flow rate was set at 0.5 mL min21. Thestarting condition was 95% solvent A (0.1% [v/v] formic acid in water) and 5%solvent B (acetonitrile) for 0.5 min, rising to 95% B at 19 min, held for 1 min,followed by 2 min of reequilibration at the starting condition. The separation wassent to an Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific)equipped with a HESI-II heated electrospray ionization source. Samples wereanalyzed in triplicate under a negative spraymode: voltage, 3,500V; capillary andauxiliary gas heater temperatures, 300°C; sheath, auxiliary, and sweep gas flowrates of 70, 10, and 5, respectively (arbitrary units). The data were acquired underfull-scan mass spectra in mass-to-charge ratio range of 100 to 600, 140,000 fullwidth at halfmaximum resolution (atmass-to-charge ratio = 200),AGC target 1e6,and maximum injection time of 200 ms.

Nuclear Morphology Phenotyping

We observed nuclear morphology in anther filament cells, which are non-pigmented and contain elongated, endopolyploid nuclei in wild-type plants(Dittmer et al., 2007). Anther filaments were fixed with ethanol:acetic acid so-lution (3:1, v/v) for 10 min, followed by exposure to 10 mg mL21 49,6-dia-midino-2-phenylindole staining solution for 15 to 30 s. Samples were immersedin distilled water for 1 min and then observed with an epifluorescence micro-scope (DM5500; Leica).

ChIP

ChIPwas performed based on the protocol fromYamaguchi et al. (2014)withthe following modifications. Anti-H3K27me3 antibodies were purchased fromAbcam (catalog no. ab6002). Vacuum infiltration of formaldehyde solution wasadjusted to 6 min followed by 4 min of incubation on ice. This procedure wasrepeated three times. Vacuum infiltration of Gly solution was modified to5 min. Sonication was applied with a Bioruptor UCD-200 (Diagenode) for 30 swith the high-intensity option followed by 30 s of pause; this 1-min cycle wasrepeated 15 times. For preclearing and immunoprecipitation capture, 45 mL ofDynabeads Protein A (ThermoFisher) was used. To purify the resulting ChIPDNA, the ChIP DNA Clean & Concentrator Kit (Zymo) was used. qPCR wasperformed as described for our RT-qPCR analysis. Primers are listed inSupplemental Table S5.

Accession Numbers

Locus identifiers of genes investigated in this study include AT1G67230(CRWN1), AT1G13220 (CRWN2), AT1G68790 (CRWN3), AT5G65770 (CRWN4),AT1G74710 (ICS1/SID2), AT2G14610 (PR1), AT3G57260 (PR2), AT1G75040(PR5), AT4G38740 (ROC1), AT5G09810 (ACT7), and AT4G18960 (AG). The RNA-seq data described here are available through NCBI (SRA BioProject IDPRJNA485018).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Mutations in Arabidopsis CRWN genes result inwidespread changes in transcript levels.


Choi et al.


http://labshare.cshl.edu/shares/mhammelllab/www-data/TEToolkit/TE_GTF/





Supplemental Figure S2. A heat map of transcript level differences of asubset of misregulated genes in different crwn genotypes underscoresthe synergy among the effects of the crwn1 and crnw2 mutations.

Supplemental Figure S3. Misregulated genes are distributed evenly acrossthe chromosome arms.

Supplemental Figure S4. Chromatin states of genes misregulated in eachcrwn mutant are shown.

Supplemental Figure S5. SA-regulated genes overlap with misregulatedgenes in crwn mutants.

Supplemental Figure S6. RNA-seq data showing expression levels of SAbiosynthesis genes and genes regulating SA biosynthesis genes.

Supplemental Figure S7. HPLC/MS data demonstrating that the sid2-1 mutation diminishes both SA and glucosylated SA levels.

Supplemental Figure S8. The sid2-1 mutation suppresses DC3000 resis-tance in crwn1 crwn2 mutants.

Supplemental Figure S9. The sid2-1 mutation partially suppresses the ro-sette dwarfism of the crwn1 crwn2 mutant, while the lengths of the boltsare not affected.

Supplemental Figure S10. crwn1 crwn2 mutants exhibit abnormal seedshape, which is not suppressed by sid2 mutations.

Supplemental Figure S11. SA overproduction does not explain the nuclearmorphology phenotypes of crwn1 crwn2 mutants.

Supplemental Figure S12. Overrepresentation of chromatin state 2 withingenes regulated in response to SA treatment.

Supplemental Figure S13. Histone H3 Lys-27 trimethylation levels at PR1are decreased in crwn1 crwn2 mutants.

Supplemental Table S1. Differential expression of TEs sorted as family incrwn mutants.

Supplemental Table S2. Differential expression of TEs sorted as individualin crwn mutants.

Supplemental Table S3. Statistical overrepresentation test for down-regulated genes in crwn mutants.

Supplemental Table S4. Statistical overrepresentation test for genesmisregulated in both crwn1 crwn2 mutants and SA-treated wild-type plants.

Supplemental Table S5. Primers used in this study.

ACKNOWLEDGMENTS

Haiyi Wang conducted the original RNA-seq experiment, and we aregrateful for her efforts to launch this project. We thank Hyong Woo Choi forhelp with the bacterial resistance tests and for providing relevant materialsfor these assays. Molly Hammell generously provided advice on the use of theTE analysis approach that her group designed. We also thank NavidMovahed, Tae Hyung Won, and Shaoqun Zhou for MS analysis and advice,as well as the Boyce Thompson Institute Computational Biology Center forguidance and bioinformatics resources. We gratefully acknowledge thehelpful comments of the anonymous referees.

Received August 16, 2018; accepted January 18, 2019; published January 29,2019.

LITERATURE CITED

Aebi U, Cohn J, Buhle L, Gerace L (1986) The nuclear lamina is a mesh-work of intermediate-type filaments. Nature 323: 560–564

Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Čech M,Chilton J, Clements D, Coraor N, Eberhard C, et al (2016) The Galaxyplatform for accessible, reproducible and collaborative biomedicalanalyses: 2016 update. Nucleic Acids Res 44: W3–W10

Al-Haboubi T, Shumaker DK, Köser J, Wehnert M, Fahrenkrog B (2011)Distinct association of the nuclear pore protein Nup153 with A- andB-type lamins. Nucleus 2: 500–509

Arabidopsis Genome Initiative (2000) Analysis of the genome sequence ofthe flowering plant Arabidopsis thaliana. Nature 408: 796–815

Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M,Marshall KA, Phillippy KH, Sherman PM, Holko M, et al (2013) NCBIGEO: Archive for functional genomics data sets—update. Nucleic AcidsRes 41: D991–D995

Bi X, Cheng YJ, Hu B, Ma X, Wu R, Wang JW, Liu C (2017) Nonrandomdomain organization of the Arabidopsis genome at the nuclear periphery.Genome Res 27: 1162–1173

Bowling SA, Clarke JD, Liu Y, Klessig DF, Dong X (1997) The cpr5 mutantof Arabidopsis expresses both NPR1-dependent and NPR1-independentresistance. Plant Cell 9: 1573–1584

Bruggeman Q, Raynaud C, Benhamed M, Delarue M (2015) To die or notto die? Lessons from lesion mimic mutants. Front Plant Sci 6: 24

Chytilova E, Macas J, Sliwinska E, Rafelski SM, Lambert GM, GalbraithDW (2000) Nuclear dynamics in Arabidopsis thaliana. Mol Biol Cell 11:2733–2741

Csoka AB, English SB, Simkevich CP, Ginzinger DG, Butte AJ, SchattenGP, Rothman FG, Sedivy JM (2004) Genome-scale expression profilingof Hutchinson-Gilford progeria syndrome reveals widespread tran-scriptional misregulation leading to mesodermal/mesenchymal defectsand accelerated atherosclerosis. Aging Cell 3: 235–243

Davidson PM, Lammerding J (2014) Broken nuclei: Lamins, nuclear me-chanics, and disease. Trends Cell Biol 24: 247–256

Dempsey DA, Vlot AC, Wildermuth MC, Klessig DF (2011) Salicylic acidbiosynthesis and metabolism. The Arabidopsis Book 9: e0156

Denais CM, Gilbert RM, Isermann P, McGregor AL, te Lindert M,Weigelin B, Davidson PM, Friedl P, Wolf K, Lammerding J (2016)Nuclear envelope rupture and repair during cancer cell migration. Sci-ence 352: 353–358

Dittmer TA, Richards EJ (2008) Role of LINC proteins in plant nuclearmorphology. Plant Signal Behav 3: 485–487

Dittmer TA, Stacey NJ, Sugimoto-Shirasu K, Richards EJ (2007) LITTLENUCLEI genes affecting nuclear morphology in Arabidopsis thaliana.Plant Cell 19: 2793–2803

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P,Chaisson M, Gingeras TR (2013) STAR: Ultrafast universal RNA-seqaligner. Bioinformatics 29: 15–21

Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBIgene expression and hybridization array data repository. Nucleic AcidsRes 30: 207–210

Egecioglu D, Brickner JH (2011) Gene positioning and expression. CurrOpin Cell Biol 23: 338–345

Fransz P, De Jong JH, Lysak M, Castiglione MR, Schubert I (2002) In-terphase chromosomes in Arabidopsis are organized as well definedchromocenters from which euchromatin loops emanate. Proc Natl AcadSci USA 99: 14584–14589

Gilford H (1904) Progeria: A form of senilism. Practitioner 73: 188–203Goldman RD, Shumaker DK, Erdos MR, Eriksson M, Goldman AE,

Gordon LB, Gruenbaum Y, Khuon S, Mendez M, Varga R, et al (2004)Accumulation of mutant lamin A causes progressive changes in nucleararchitecture in Hutchinson-Gilford progeria syndrome. Proc Natl AcadSci USA 101: 8963–8968

Grob S, Schmid MW, Grossniklaus U (2014) Hi-C analysis in Arabidopsisidentifies the KNOT, a structure with similarities to the flamenco locusof Drosophila. Mol Cell 55: 678–693

Gruenbaum Y, Foisner R (2015) Lamins: Nuclear intermediate filamentproteins with fundamental functions in nuclear mechanics and genomeregulation. Annu Rev Biochem 84: 131–164

Gu Y, Zebell SG, Liang Z, Wang S, Kang BH, Dong X (2016) Nuclear porepermeabilization is a convergent signaling event in effector-triggeredimmunity. Cell 166: 1526–1538.e11

Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, Talhout W, EussenBH, de Klein A, Wessels L, de Laat W, et al (2008) Domain organizationof human chromosomes revealed by mapping of nuclear lamina inter-actions. Nature 453: 948–951

Guo T, Mao X, Zhang H, Zhang Y, Fu M, Sun Z, Kuai P, Lou Y, Fang Y(2017) Lamin-like proteins negatively regulate plant immunity through NACWITH TRANSMEMBRANE MOTIF1-LIKE9 and NONEXPRESSOR OF PRGENES1 in Arabidopsis thaliana. Mol Plant 10: 1334–1348

Guo Y, Kim Y, Shimi T, Goldman RD, Zheng Y (2014) Concentration-dependent lamin assembly and its roles in the localization of other nu-clear proteins. Mol Biol Cell 25: 1287–1297






















Guy L, Kultima JR, Andersson SG (2010) genoPlotR: Comparative geneand genome visualization in R. Bioinformatics 26: 2334–2335

Harr JC, Luperchio TR, Wong X, Cohen E, Wheelan SJ, Reddy KL (2015)Directed targeting of chromatin to the nuclear lamina is mediated bychromatin state and A-type lamins. J Cell Biol 208: 33–52

Harr JC, Gonzalez-Sandoval A, Gasser SM (2016) Histones and histonemodifications in perinuclear chromatin anchoring: From yeast to man.EMBO Rep 17: 139–155

Heberle H, Meirelles GV, da Silva FR, Telles GP, Minghim R (2015) In-teractiVenn: A web-based tool for the analysis of sets through Venndiagrams. BMC Bioinformatics 16: 169

Hutchinson J (1886) Congenital absence of hair and mammary glands withatrophic condition of the skin and its appendages, in a boy whosemother had been almost wholly bald from alopecia areata from the ageof six. Med Chir Trans 69: 473–477

Ikegami K, Egelhofer TA, Strome S, Lieb JD (2010) Caenorhabditis ele-gans chromosome arms are anchored to the nuclear membrane viadiscontinuous association with LEM-2. Genome Biol 11: R120

Jin Y, Tam OH, Paniagua E, Hammell M (2015) TEtranscripts: A packagefor including transposable elements in differential expression analysis ofRNA-seq datasets. Bioinformatics 31: 3593–3599

Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013)TopHat2: Accurate alignment of transcriptomes in the presence of in-sertions, deletions and gene fusions. Genome Biol 14: R36

Kühlbrandt W (2015) Structure and function of mitochondrial membraneprotein complexes. BMC Biol 13: 89

Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast andmemory-efficient alignment of short DNA sequences to the human ge-nome. Genome Biol 10: R25

Lombardi ML, Lammerding J (2011) Keeping the LINC: The importance ofnucleocytoskeletal coupling in intracellular force transmission and cel-lular function. Biochem Soc Trans 39: 1729–1734

Love MI, Huber W, Anders S (2014) Moderated estimation of foldchange and dispersion for RNA-seq data with DESeq2. Genome Biol15: 550

Masuda K, Takahashi S, Nomura K, Arimoto M, Inoue M (1993) Residualstructure and constituent proteins of the peripheral framework of thecell-nucleus in somatic embryos from Daucus carota L. Planta 191:532–540

Masuda K, Xu ZJ, Takahashi S, Ito A, Ono M, Nomura K, Inoue M (1997)Peripheral framework of carrot cell nucleus contains a novel proteinpredicted to exhibit a long alpha-helical domain. Exp Cell Res 232:173–181

Meaburn KJ, Cabuy E, Bonne G, Levy N, Morris GE, Novelli G, Kill IR,Bridger JM (2007) Primary laminopathy fibroblasts display altered ge-nome organization and apoptosis. Aging Cell 6: 139–153

Meier I (2016) LINCing the eukaryotic tree of life: Towards a broad evo-lutionary comparison of nucleocytoplasmic bridging complexes. J CellSci 129: 3523–3531

Meier I, Griffis AH, Groves NR, Wagner A (2016) Regulation of nuclearshape and size in plants. Curr Opin Cell Biol 40: 114–123

Meier I, Richards EJ, Evans DE (2017) Cell biology of the plant nucleus.Annu Rev Plant Biol 68: 139–172

Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD(2017) PANTHER version 11: Expanded annotation data from GeneOntology and Reactome pathways, and data analysis tool enhance-ments. Nucleic Acids Res 45: D183–D189

Mikulski P, Hohenstatt ML, Farrona S, Smaczniak C, Kaufmann K,Angenent G, Schubert D (2017) PWWP INTERACTOR OF POLY-COMBS (PWO1) links PcG-mediated gene repression to the nuclearlamina in Arabidopsis. bioRxiv, doi:10.1101/220541

Moeder W, Yoshioka K (2008) Lesion mimic mutants: A classical, yet stillfundamental approach to study programmed cell death. Plant SignalBehav 3: 764–767

Nawrath C, Métraux JP (1999) Salicylic acid induction-deficient mutants ofArabidopsis express PR-2 and PR-5 and accumulate high levels of ca-malexin after pathogen inoculation. Plant Cell 11: 1393–1404

Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I,Brugman W, Gräf S, Flicek P, Kerkhoven RM, van Lohuizen M, et al(2010) Molecular maps of the reorganization of genome-nuclear laminainteractions during differentiation. Mol Cell 38: 603–613

Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, van SteenselB (2006) Characterization of the Drosophila melanogaster genome at thenuclear lamina. Nat Genet 38: 1005–1014

Poulet A, Duc C, Voisin M, Desset S, Tutois S, Vanrobays E, Benoit M,Evans DE, Probst AV, Tatout C (2017) The LINC complex contributes toheterochromatin organisation and transcriptional gene silencing inplants. J Cell Sci 130: 590–601

Reddy KL, Zullo JM, Bertolino E, Singh H (2008) Transcriptional repres-sion mediated by repositioning of genes to the nuclear lamina. Nature452: 243–247

Rivas-San Vicente M, Plasencia J (2011) Salicylic acid beyond defence: Itsrole in plant growth and development. J Exp Bot 62: 3321–3338

Robert-Seilaniantz A, Grant M, Jones JDG (2011) Hormone crosstalk inplant disease and defense: More than just jasmonate-salicylate antago-nism. Annu Rev Phytopathol 49: 317–343

Sakamoto Y, Takagi S (2013) LITTLE NUCLEI 1 and 4 regulate nuclearmorphology in Arabidopsis thaliana. Plant Cell Physiol 54: 622–633

Scaffidi P, Misteli T (2006) Lamin A-dependent nuclear defects in humanaging. Science 312: 1059–1063

Schreiber KH, Kennedy BK (2013) When lamins go bad: Nuclear structureand disease. Cell 152: 1365–1375

Sequeira-Mendes J, Aragüez I, Peiró R, Mendez-Giraldez R, Zhang X,Jacobsen SE, Bastolla U, Gutierrez C (2014) The functional topographyof the Arabidopsis genome is organized in a reduced number of linearmotifs of chromatin states. Plant Cell 26: 2351–2366

Seyfferth C, Tsuda K (2014) Salicylic acid signal transduction: The initia-tion of biosynthesis, perception and transcriptional reprogramming.Front Plant Sci 5: 697

Stingl N, Krischke M, Fekete A, Mueller MJ (2013) Analysis of defensesignals in Arabidopsis thaliana leaves by ultra-performance liquidchromatography/tandem mass spectrometry: Jasmonates, salicylic acid,abscisic acid. Methods Mol Biol 1009: 103–113

Stroud H, Greenberg MV, Feng S, Bernatavichute YV, Jacobsen SE (2013)Comprehensive analysis of silencing mutants reveals complex regula-tion of the Arabidopsis methylome. Cell 152: 352–364

Tamura K, Fukao Y, Hatsugai N, Katagiri F, Hara-Nishimura I (2017)Nup82 functions redundantly with Nup136 in a salicylic acid-dependentdefense response of Arabidopsis thaliana. Nucleus 8: 301–311

Tapley EC, Starr DA (2013) Connecting the nucleus to the cytoskeleton bySUN-KASH bridges across the nuclear envelope. Curr Opin Cell Biol 25:57–62

Tatout C, Evans DE, Vanrobays E, Probst AV, Graumann K (2014) Theplant LINC complex at the nuclear envelope. Chromosome Res 22:241–252

Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ,Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly andquantification by RNA-Seq reveals unannotated transcripts and isoformswitching during cell differentiation. Nat Biotechnol 28: 511–515

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H,Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcriptexpression analysis of RNA-seq experiments with TopHat and Cuf-flinks. Nat Protoc 7: 562–578

van Steensel B, Belmont AS (2017) Lamina-associated domains: Links withchromosome architecture, heterochromatin, and gene repression. Cell169: 780–791

van Wees S (2008) Phenotypic analysis of Arabidopsis mutants: Trypanblue stain for fungi, oomycetes, and dead plant cells. CSH Protoc 2008:pdb prot4982

Vlot AC, Dempsey DA, Klessig DF (2009) Salicylic acid, a multifacetedhormone to combat disease. Annu Rev Phytopathol 47: 177–206

Wang H, Dittmer TA, Richards EJ (2013) Arabidopsis CROWDED NU-CLEI (CRWN) proteins are required for nuclear size control and heter-ochromatin organization. BMC Plant Biol 13: 200

Wang Q, Liu S, Lu C, La Y, Dai J, Ma H, Zhou S, Tan F, Wang X, Wu Y,et al (2019) Roles of CRWN-family proteins in protecting genomic DNAagainst oxidative damage. J Plant Physiol 233: 20–30

Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismatesynthase is required to synthesize salicylic acid for plant defence. Nature414: 562–565

Yamaguchi N, Winter CM, Wu MF, Kwon CS, William DA, Wagner D(2014) PROTOCOLS: Chromatin immunoprecipitation from Arabidopsistissues. The Arabidopsis Book 12: e0170


Choi et al.



Zhang X, Clarenz O, Cokus S, Bernatavichute YV, Pellegrini M, GoodrichJ, Jacobsen SE (2007) Whole-genome analysis of histone H3 lysine 27trimethylation in Arabidopsis. PLoS Biol 5: e129

Zheng XY, Zhou M, Yoo H, Pruneda-Paz JL, Spivey NW, Kay SA, Dong X(2015) Spatial and temporal regulation of biosynthesis of the plant im-mune signal salicylic acid. Proc Natl Acad Sci USA 112: 9166–9173

Zheng X, Hu J, Yue S, Kristiani L, Kim M, Sauria M, Taylor J, Kim Y,Zheng Y (2018) Lamins organize the global three-dimensional genomefrom the nuclear periphery. Mol Cell 71: 802–815.e7

Zhou M, Wang W, Karapetyan S, Mwimba M, Marqués J, Buchler NE,Dong X (2015) Redox rhythm reinforces the circadian clock to gate im-mune response. Nature 523: 472–476





Loss of CRWN Nuclear Proteins Induces Cell Death and ... · nents, based on pioneering studies by...

Documents

Transcript of Loss of CRWN Nuclear Proteins Induces Cell Death and ... · nents, based on pioneering studies by...